Microbiological analyses of sediments located near a point source for petrogenic chemicals resulted in the isolation of a pyrene-mineralizing bacterium. This isolate was identified as a Mycobacterium sp. on the basis of its cellular and colony morphology, gram-positive and strong acid-fast reactions, diagnostic biochemical tests, 66.6% G+C content of the DNA, and high-molecular-weight mycolic acids (C58 to C64). The mycobacterium mineralized pyrene when grown in a mineral salts medium supplemented with nutrients but was unable to utilize pyrene as a sole source of carbon and energy. The mycobacterium grew well at 24 and 30°C and minimally at 35°C. No growth was observed at 5 or 42°C. The mycobacterium grew well at salt concentrations up to 4%. Pyrene-induced Mycobacterium cultures mineralized 5% of the pyrene after 6 h and reached a maximum of 48% mineralization within 72 h. Treatment of induced and noninduced cultures with chloramphenicol showed that pyrene-degrading enzymes were inducible in this Mycobacterium sp. This bacterium could also mineralize other polycyclic aromatic hydrocarbons and alkyl-and nitro-substituted polycyclic aromatic hydrocarbons including naphthalene, phenanthrene, fluoranthene, 3-methylcholanthrene, 1-nitropyrene, and 6-nitrochrysene. This is the first report of a bacterium able to extensively mineralize pyrene and other polycyclic aromatic hydrocarbons containing four aromatic rings.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.

Human intestinal microbial flora were screened for their abilities to reduce nitroaromatic compounds by growing them on brain heart infusion agar plates containing 1-nitropyrene. Bacteria metabolizing 1-nitropyrene, detected by the appearance of clear zones around the colonies, were identified as Clostridium leptum, Clostridium paraputrificum, Clostridium clostridiiforme, another Clostridium sp., and a Eubacterium sp. These bacteria produced aromatic amines from nitroaromatic compounds, as shown by thin-layer chromatography, high-pressure liquid chromatography, and biochemical tests. Incubation of three of these bacteria with 1nitropyrene, 1,3-dinitropyrene, and 1,6-dinitropyrene inactivated the direct-acting mutagenicity associated with these compounds. Menadione and o-iodosobenzoic acid inhibited nitroreductase activity in all of the isolates, indicating the involvement of sulfhydryl groups in the active site of the enzyme. The optimum pH for nitroreductase activity was 8.0. Only the Clostridium sp. required added flavin adenine dinucleotide for nitroreductase activity. The nitroreductases were constitutive and extracellular. An activity stain for the detection of nitroreductase on anaerobic native polyacrylamide gels was developed. This activity stain revealed only one isozyme in each bacterium but showed that the nitroreductases from different bacteria had distinct electrophoretic mobilities.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295-297, 1975), was previously identified as Beijerinckiu sp. strain B1. In this investigation, 16s rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckiu sp. strain Bl. The sequence of the 16s rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.A strain designated Beijerinckia sp. strain B1 was originally isolated for its ability to grow with biphenyl as a carbon and energy source (6). This organism can utilize biphenyl, naphthalene, and phenanthrene as sole sources of carbon and energy and co-oxidize a wide variety of polycyclic aromatic hydrocarbons to carbon dioxide and mixtures of ring fission products (1, 6, 8, 9, 12). Biphenyl-induced cells of a mutant of this strain, B8/36, oxidize biphenyl, anthracene, phenanthrene, benzo[a]pyrene, and benz [a]anthracene to cis-dihydrodiols (1, 6, 8, 9, 12, 17). The metabolic pathway for the degradation of benz [a]anthracene is inducible by biphenyl, salicylate, and mxylene (12). Because of the wide range of polycyclic aromatic hydrocarbons degraded by this organism, extensive genetic and metabolic pathway studies have been done (1, 3, 6, 8, 9). Molecular analysis of the enzymes involved in aromatic degradation has been reported by Kim and Zylstra (10).Since strain B1 has potential use in the bioremediation of polycyclic aromatic hydrocarbon-contaminated environmental sites, we reevaluated the identification of Beijerinckia sp. strain B1 by using biochemical tests, DNA-DNA hybridization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) soluble protein analysis, fatty acid methyl ester (FAME) analysis, and 16s rRNA gene sequence analysis. FAME analysis. Bacterial cultures were harvested from Trypticase soy broth-5% sheep blood agar for total cellular fatty acid analysis. Fatty acids were extracted by following the Microbial Identification System (MIDI; Microbial ID, Inc., Newark, Del.) instructions. MATERIALS AND METHODSPreparation of soluble protein fractions. The bacterial strains listed above were grown on Trypticase soy broth-5% sheep blood agar plates for 16 h at 30°C. Several colonies were combined in 300 p1 of Tris-HC1 (20 mM, pH 6.8) and sonicated for 4 30-s pulses at maximum power with 30-s cooling intervals in an ice bath. Unbroken cells and large fragm...

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